The proteasome inhibitors bortezomib (Btz), carfilzomib (Cfz) and ixazomib (Ixz) are successfully used for the treatment of multiple myeloma (MM), but lack clinical efficacy against solid tumors despite demonstrating significant activity in tissue culture and animal models. In an effort to understand this discrepancy, we decided to focus on triple-negative breast cancers (TNBC), a genetically diverse group that lacks targeted treatments. TNBCs overexpress pro-apoptotic protein NOXA, which plays a key role in proteasome inhibitor-induced apoptosis, and are proteasome-addicted. The proteasome has three pairs of active sites: 5, 1, and 2. The primary target of Btz and Cfz are the 5 sites. At concentrations of Btz and Cfz that induce apoptosis in TNBC in culture, co-inhibition of either 1 or 2 is also observed. These concentrations exceed clinically relevant concentrations, potentially explaining why proteasome inhibitors have no in vivo activity in solid tumors, and suggesting that stronger co-inhibition of either 1 or 2 would improve in vivo activity. It is not known whether the 2 or the 1 site is a better target. We developed specific inhibitors of the 1 and 2 sites and CRISPR- generated mutations of these sites. We found that LU-102 and mutations in the 2 sites are much stronger sensitizers of TNBC cells to Cfz and Btz than the 1 inhibitor NC-021 or 1 mutations; 2 mutations sensitize orthotopic xenografts of TNBC cells to sub-toxic doses of Cfz. LU-102 and 2 mutations block recovery of proteasome activity in Cfz-treated cells. In contrast to myeloma cells, proteasome activity recovers in TNBC cells treated with clinically relevant concentrations of Cfz. We found that LU-102 and 2 mutations inhibit recovery of proteasome activity by causing aggregation of Nrf1 (NFE2L1/TCF11), a transcription factor that up- regulates proteasome genes expression in cells treated with 5 inhibitors. NC-021 and 1 mutations do not block recovery of proteasome activity. Therefore, we hypothesize that 2 inhibitors will sensitize TNBCs to FDA-approved 5 inhibitors, by the mechanism which involves tumor-specific inhibition of Nrf1-mediated recovery of proteasome activity. We further hypothesize that targeting enzymes involved in Nrf1 activation will also sensitize TNBC to proteasome inhibitors. The specific aims of this proposal are: (i) to determine whether 2 inhibitors sensitize TNBC to Cfz, Btz, and Ixz in vivo; (ii) to determine whether 2 inhibitors block recovery of proteasome activity in a tumor-specific fashion in vivo and determine whether alternative approaches to target this recovery sensitize tumors to FDA-approved inhibitors. Accomplishing these aims will demonstrate that the 2 sites and Nrf1 are effective drug targets in TNBC, establish the mechanism of action of 2 inhibitors, pave the road to clinical trials of 2 inhibitors in TNBC, and launch future work in solid tumors of different organs.